Organic acid synthesis from C1 substrates

ABSTRACT

Presented herein are biocatalysts and methods for converting C1-containing materials to organic acids such as muconic acid or adipic acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Non-provisional application Ser. No. 15/252,648, filed Aug. 31, 2016, which claims priority to, and the benefit of, U.S. Provisional Application No. 62/212,264, filed Aug. 31, 2015, the contents of which are incorporated by reference in their entirety.

CONTRACTUAL ORIGIN

The United States Government has rights in this invention under Contract No. DE-AC36-08GO28308 between the United States Department of Energy and Alliance for Sustainable Energy, LLC, the Manager and Operator of the National Renewable Energy Laboratory.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronic text file entitled “15-92_ST25.txt,” having a size in bytes of 224 kb and created on Aug. 22, 2016. Pursuant to 37 CFR § 1.52(e)(5), the information contained in the above electronic file is hereby incorporated by reference in its entirety.

BACKGROUND

Methane is a critical component of Earth's carbon cycle that contributes 18% to the Earth's warming. It is the major constituent of natural gas and biogas, composing up to 80% of the latter. CH₄ is emitted from a variety of natural and anthropogenic sources. Human-related activities, such as fossil fuel production (e.g., underground coal mining, oil and gas production), agriculture (e.g., enteric fermentation in livestock, manure management, and rice cultivation), landfills, and municipal wastewater are major contributors of global CH₄ emission. Anthropogenic CH₄ emission accounts for more than 60% of the total CH₄ budget (≈300 tg yr⁻¹).

CH₄ is not only one of the major contributors for climate change, it is also the primary target for near-term climate regulation. Stranded natural gas (SNG), oil-associated gas and almost all waste-derived biogas are not economically feasible sources of energy due to small size or remote location. Each year, up to 116 million tonnes of oil-associated methane and 40 million tonnes of biogas (equivalent to 30% of the total US transportation fuel) are flared, which represents lost energy (five quadrillion BTU of fossil fuel energy), unnecessary greenhouse gas emissions and dangerous air pollution. Strategies for effectively converting CH₄ into valuable compounds offer promising new technologies for global warming stabilization and possibly reduction. Methane-rich biogas offers a renewable alternative to fossil natural gas as a feedstock and intermediate in bioprocesses, adding to our capacity for biofuels and biobased products to supplement those available from lignocellulosics or algae.

The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.

SUMMARY

The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods that are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements.

Provided herein are engineered cells that are able to convert a C1 substrate to an organic acid and that comprise one or more exogenously added genes encoding a 3-dehydroshikimate dehydratase, a protocatechuic acid decarboxylase, a catechol 1,2-dioxygenase, aroG^(fbr), trpE^(fbr), a phosphoenolpyruvate synthase, a transketolase, a phosphoketolase, and/or a RuBisCO polypetide.

In some embodiments, the engineered cells comprise AroZ from Klebsiella variicola, AroY from Enterobacter cloacae, and/or CatA from Acinetobacter.

In various embodiments, the phosphoketolase is PktA or PktB and/or the RuBisCO polypetide is CbbL, CbbS, CbbQ and/or CbbP.

In certain embodiments, the engineered cell is a C1 metabolizing cell, such as cells from the genera Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocystis, Methylomicrobium, Methanomonas, Methylophilus, Methylobacillus, Methylobacterium, Hyphomicrobium, Xanthobacter, Bacillus, Paracoccus, Nocardia, Arthrobacter, Rhodopseudomonas, Candida, Yarrowia, Hansenula, Pichia, Torulopsis, Rhodotorula or Pseudomonas; and/or methanotrophic bacterial cells or a methylotrophic (methanol-utilizing) bacterial or yeast cells.

In some embodiments, the C1 metabolizing cell is a methanotroph or methylotroph; is from an organism from the genus Methylobacterium, Methylomicrobium, or Methylococcus (such as Methylobacterium extorquens, Methylobacterium radiotolerans, Methylobacterium populi, Methylobacterium chloromethanicum, Methylobacterium nodulans, Methylomicrobium alkaliphilum or a combination thereof); or is M. alkaliphilum 20Z or M. buryatense.

In various embodiments, the C1 substrate is methane, methanol, carbon dioxide, carbon monoxide, syngas, natural gas, or biogas; and/or the organic acid is muconic acid or adipic acid.

Also provided are methods for producing an organic acid by culturing cells described herein with a C1 substrate and recovering the organic acid from the culture.

In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting.

FIG. 1 shows the nucleic acid sequence (A; SEQ ID NO:1) and amino acid sequence (B; SEQ ID NO:2) of 3-dehydroshikimate dehydratase from Podospora anserina.

FIG. 2 shows the nucleic acid sequence (A; SEQ ID NO:11) and amino acid sequence (B; SEQ ID NO:12) of protocatechuic acid decarboxylase from Enterobacter cloacae.

FIG. 3 shows the nucleic acid sequence (A; SEQ ID NO:15) and amino acid sequence (B; SEQ ID NO:16) of catechol 1,2-dioxygenase from Candida albicans.

FIG. 4 shows the nucleic acid sequence (A; SEQ ID NO:33) and amino acid sequence (B; SEQ ID NO:34) of aroG^(fbr) from E. coli codon optimized for expression in Methylomicrobium alcaliphilum.

FIG. 5 shows the nucleic acid sequence (A; SEQ ID NO:35) and amino acid sequence (B; SEQ ID NO:36) of trpE^(fbr) from E. coli codon optimized for expression in Methylomicrobium alcaliphilum.

FIG. 6 shows the nucleic acid sequence (A; SEQ ID NO:37) and amino acid sequence (B; SEQ ID NO:38) of phosphoenolpyruvate synthase from Methylomicrobium alcaliphilum 20Z.

FIG. 7 shows the nucleic acid sequence (A; SEQ ID NO:39) and amino acid sequence (B; SEQ ID NO:40) of transketolase from Methylomicrobium alcaliphilum 20Z.

FIG. 8 shows a synthetic pathway for muconate biosynthesis from methane. E4P, erythrose 4-phosphate; PEP, phosphoenolpyruvate; DAHP, 3-deoxy-d-arabino-heptulosonate-7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; (1), DAHP synthase; (2), DHQ synthase; (3), DHQ dehydratase; (4), Shikimate dehydrogenase; (5), 3-dehydroshikimate dehydratase; (6), protocatechuate decarboxylase; (7), catechol 1,2-dioxygenase.

FIG. 9 shows a diagram of a vector useful for constitutive expression of genes such as AroZ, AroY and CatA in Methylomicrobium alcaliphilum 20Z.

FIG. 10 shows gene expression levels of cells engineered to express 3-dehydroshikimate dehydratase (AroZ), protocatechuic acid decarboxylase (AroY) and catechol 1,2-dioxygenase (CatA).

FIG. 11 shows a diagram of a vector for expression of genes for enhanced flux to muconic acid pathway precursors.

FIG. 12 shows a diagram of the pCAH01 inducible expression vector. Knr (kanamycin resistance), Ampr (ampicillin resistance), tetR (transcriptionally-fused tetracycline repressor), OriV/OriT (IncP-based origin of replication/transfer), trfA (OriV replication initiation protein).

FIG. 13 shows growth (OD₆₀₀) and cis,cis-muconic acid (ccMA) production for Methylomicrobium alcaliphilum 20Z cells engineered express 3-dehydroshikimate dehydratase (AroZ), protocatechuic acid decarboxylase (AroY) and catechol 1,2-dioxygenase (CatA).

FIG. 14 shows increased carbon conversion efficiency from biocatalysts overexpressing the phosphoketolase enzyme (pktA and pktB).

FIG. 15 shows the Embden-Meyerhof-Parnas (EMP) pathway, the phosphoketolase pathway (PKT) and how each acts on fructose-6-phosphate (F6P).

FIG. 16 shows an exemplary plasmid for high, constitutive expression of genes (such as Pkt isoforms PktA and PktB) from the methanol dehydrogenase promoter (Pmxa).

FIG. 17 shows (A) relative Pkt expression in wild-type (WT) M. buryatense cells or cells expressing plasmids containing PktA or PktB as determined by real-time PCR analysis; (B) SDS-PAGE analysis of whole-cell lysates demonstrating increased Pkt expression by engineered strains; and (C) in vitro conversion of fructose-6-phosphate (F6P) to acetyl-phosphate (AcP) by whole-cell lysates from wild-type (WT) cells or cells expressing plasmids containing PktA or PktB.

FIG. 18 shows that Pkt overexpression increases carbon conversion efficiency (CCE) by (A) real-time CH₄ consumption (off gas analysis) for wild-type M. buryatense cells (WT; upper line) and Pkt-expressing cells (pPKT; lower line); (B) cell growth (DCW, dry cell weight) as measured over time in a 0.5 L gas bioreactor with continuous CH₄ feed (20% CH₄ in air); and (C) the ratio of biomass DCW to total CH₄ consumption after 96 hours.

FIG. 19 shows an exemplary vector for heterologous expression of the autotrophic pathway. The cbbL, cbbS, and cbbQ genes encoding the subunit and regulatory proteins of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoribulokinase-encoding cbbP from Methylococcus capsulatus are included in the vector.

DETAILED DESCRIPTION

Presented herein are methods and biocatalysts for biological conversion of C1 substrates such as methane, methanol, and carbon-dioxide (or materials containing these compounds such as biogas) into organic acids such as muconic acid or adipic acid. Methanotrophs do not natively produce muconic acid, but disclosed herein are processes for altering methanotrophs to enable the production of organic acids such as muconic acid or adipic acid from C1 (e.g., methane) containing sources.

The cells of methanotrophic organisms may be modified to express one or more exogenously added genes encoding enzymes that allow the cell to convert C1 substrates such as methane to muconic acid or adipic acid. Exemplary enzymes include 3-dehydroshikimate dehydratase, protocatechuic acid decarboxylase and catechol 1,2-dioxygenase enzymes. Specific examples include the enzymes 3-dehydroshikimate dehydratase (AroZ) from Podospora anserina, protocatechuic acid decarboxylase (AroY) from Enterobacter cloacae, and catechol 1,2-dioxygenase (CatA) from Candida albicans, the nucleic acid and amino acid sequences of which are provided in FIG. 1-3. Functional homologs of these enzymes from other species are also suitable for use in the present disclosure.

Additional suitable examples of 3-dehydroshikimate dehydratases include those from Klebsiella variicola (SEQ ID NOS:3 and 4), Bacillus thuringiensis (SEQ ID NOS:5 and 6), Enterobacter aerogenes (SEQ ID NOS:7 and 8), and Acinetobacter baylyi (SEQ ID NOS:9 and 10). An additional exemplary protocatechuic acid decarboxylase is the AroY gene from Klebsiella variicola (SEQ ID NOS:13 and 14). Additional suitable examples of catechol 1,2-dioxygenases include the CatA gene from Pseudomonas putida (SEQ ID NOS:19 and 20) and the CatA gene from Acinetobacter spp. (SEQ ID NOS:17 and 18) as well as a variant version of this gene where the proline residue at position 76 is mutated to an alanine.

The cells may also be engineered to express one or more exogenously added genes encoding aroG^(fbr), trpE^(fbr), a phosphoenolpyruvate synthase, or a transketolase. The designation “fbr” refers to feedback resistant variants of the indicated enzymes. Suitable examples include aroG^(fbr) from E. coli, trpE^(fbr) from E. coli, phosphoenolpyruvate synthase from Methylomicrobium alcaliphilum 20Z or transketolase from Methylomicrobium alcaliphilum 20Z, the nucleic acid and amino acid sequences of which are provided in FIG. 4-7. Functional homologs of these enzymes from other species are also suitable for use in the present disclosure. All nucleic acid sequences may be codon optimized for expression in the host cell.

Muconic acid is a dicarboxylic acid that may be converted via hydrogenation into adipic acid with high yield and specificity. Currently, nylon-6,6 accounts for greater than 85% of global adipic acid production. The dicarboxylic functionality of adipic acid affords a wide variety of upgrading strategies including polymerization, lactonization, diolization, and ketonization. As such, adipic acid can be readily converted to other large-market, high-value molecules and fuel precursors, such as plasticizers, lubricants, engineering resins, polyurethanes, and food gelatin. Additionally, muconic acid itself can be upgraded to produce commodity chemicals beyond adipic acid, including terepthalic and trimellitic acids. Conventional adipic acid production is almost exclusively dependent on petroleum-derived feedstocks and accounts for nearly 10% of global N₂O emissions. Thus, the production of muconic acid or adipic acid through a biocatalytic upgrading process from renewable methane sources offers a novel production route with significant economic and sustainability benefits. Furthermore, the diversity of products that can be made from adipic acid assures a large demand for the heretofore poorly valorized biogas.

Microbial utilization of CH₄ (methanotrophy) can occur in both aerobic and anaerobic environments, but only aerobic methanotrophic bacteria (methanotrophs) have been isolated in pure culture. Methanotrophs use methane monooxygenase for the first oxidation step that converts CH₄ into methanol (CH₃OH), which is further oxidized to formaldehyde (CH₂O). Gammaproteobacteria, such as M. alcaliphilum 20Z, assimilate formaldehyde through the assimilatory ribulose monophosphate (RuMP) pathway. The first part of the pathway is the condensation of CH₂O with ribulose-5-phosphate (Ru5P) to produce 3-hexulose-6-phosphate, which is subsequently isomerized to fructose-6-phosphate.

Muconic acid production naturally occurs during the catabolism of aromatic compounds in a limited number of microorganisms. Production of muconic acid in various microbes has been achieved by exploiting naturally occurring intermediary metabolic pathways involved in the detoxification or catabolism of aromatic compounds such as toluene and benzoate. Methanotrophs do not naturally possess a pathway to produce muconic acid, and are obligate C1-utilizers. As such, a novel metabolic route to muconic acid biosynthesis must be employed in order to leverage the innate capacity of methanotrophic biocatalysts.

Shunting naturally occurring metabolites toward muconic acid presents a direct approach for synthesizing this product from renewable feedstocks. The shikimate pathway is involved in the synthesis of aromatic amino acids, and muconic acid can be synthesized in a three step process from the 3-dehydroshikimate (DHS) intermediate produced in this pathway (FIG. 8). Alternatively, muconic acid can be generated by converting the anthranilate intermediate of tryptophan biosynthesis into catechol by the anthranilate 1,2 dioxygenase.

Methylomicrobium spp. do not require exogenous amino acids for growth, indicating that the up-front metabolic machinery is already in place. Genomic analysis of M. alkaliphilum 20Z confirmed all genes of the shikimate and pentose phosphate (non-oxidative, PPP) pathways are present, verifying the ability to produce DHS. Further, the strain has high flux via PPP and a high pool of the key precursors for the proposed route of muconate biosynthesis, highlighting the feasibility of engineering muconate production in the organism.

Synthetic design of a three-step muconate biosynthesis pathway may be established and optimized in bacteria (e.g., P. putida) and yeast, using aromatics and glucose as precursors for muconic acid biosynthesis, respectively. Alternatively, muconate biosynthesis may be connected to methane precursors. In one specific example, a three-step synthetic pathway comprised of the enzymes 3-dehydroshikimate dehydratase from Podospora anserina, protocatechuic acid decarboxylase from Enterobacter cloacae, and catechol 1,2-dioxygenase from Candida albicans may be incorporated into M. alcaliphilum 20Z. The genes may be codon optimized for 20Z and can be used in the construction of both integrative and replicative plasmids.

Suitable cells include those able to convert a C1 substrate to an organic acid or a C1 metabolizing cell, including methanotrophic bacterial cells or methylotrophic (methanol-utilizing) bacterial or yeast cells. In certain embodiments, the cell is a methanotroph or methylotroph.

Examples include cells from the genera Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocystis, Methylomicrobium, Methanomonas, Methylophilus, Methylobacillus, Methylobacterium, Hyphomicrobium, Xanthobacter, Bacillus, Paracoccus, Nocardia, Arthrobacter, Rhodopseudomonas, Candida, Yarrowia, Hansenula, Pichia, Torulopsis, Rhodotorula or Pseudomonas. Specific examples include cells from Methylobacterium extorquens, Methylobacterium radiotolerans, Methylobacterium populi, Methylobacterium chloromethanicum, Methylobacterium nodulans, Methylomicrobium buryatense, Methylomicrobium alkaliphilum or a combination thereof. In some embodiments, the cell is M. alkaliphilum 20Z.

In various embodiments, the methanotroph is a Methylomonas sp. 16a (ATCC PTA 2402), Methylosinus trichosporium (NRRL B-1 1,196), Methylosinus sporium (NRRL B-1 1,197), Methylocystis parvus (NRRL fil 1, 198), Methylomonas methanica (NRRL B-1 1,199), Methylomonas albus (NRRL fil 1, 200), Methylobacter capsulatus (NRRL B-1 1,201), Methylobacterium organophilum (ATCC 27,886), Methylomonas sp. AJ-3670 (FERM P-2400), Methylocella silvestris, Methylacidiphilum infernorum, Methylibium petroleiphilum, or a combination thereof.

In certain embodiments, the organic acid may be muconic acid or adipic acid. The C1 substrate may be a C1 compound such as methane, methanol, carbon dioxide, or carbon monoxide; or a C1-containing material such as syngas, natural gas, or biogas; or materials comprising any of these compounds or materials. Biogas, such as that generated from anaerobic digestion of waste streams, including wastewater derived from conventional biorefineries, is one example of a versatile, renewable C1-containing material source. Currently, biogas is utilized for on-site heat and energy production or sold into the national grid for minimal compensation. However, microbial conversion of biogas to value-added chemicals using the modified microorganisms disclosed herein offers immense valorization potential.

These biocatalysts hold great promise for the capture and conversion of methane from anaerobic-digestion-derived biogas and other anthropogenic CH₄ sources. However, biogas contains more than just methane, with CO₂ typically comprising about 40% of biogas streams. A biocatalyst capable of co-utilization and conversion of CO₂ and CH₄ allows for complete utilization of biogas streams and, in turn, enhanced carbon conversion efficiencies. Such a biocatalyst may also shift the landscape of greenhouse gas mitigation, capture, and conversion pursuits, providing a novel, photosynthesis-independent CO₂ biocatalyst.

The biocatalyst microorganisms described herein may be further modified to express or overexpress a ribulose 1,5 bisphosphate carboxylase/oxygenase (RuBisCO) enzyme that can allow autotrophic growth in the presence an energy source such as methane, formate or hydrogen). Both homologous and heterologous overexpression of RuBisCO enzymes may increase CO₂ utilization in an array of methanotrophs, including those that do not encode genes that enable autotrophic growth.

Exemplary enzymes include the cbbL, cbbS and cbbQ genes encoding the subunit and regulatory proteins of RuBisCO, along with the phosphoribulokinase-encoding cbbP. For example, the nucleotide and amino acid sequences for cbbL, cbbS, cbbQ and cbbP from Methylococcus capsulatus are depicted as SEQ ID NOS:25 and 26 (cbbL); SEQ ID NOS:27 and 28 (cbbS); SEQ ID NOS:29 and 30 (cbbQ); and SEQ ID NOS:31 and 32 (cbbP), respectively. Functional homologs of these enzymes from other species are also suitable for use in the present disclosure.

Additional embodiments include biocatalysts engineered to express or overexpress one or more genes encoding a phosphoketolase enzyme. Phosphoketolases catalyze the phosphorylytic cleavage of xylulose-5-phosphate to acetyl-phosphate and glyceraldehyde-3-phosphate or fructose-6-phosphate to acetyl-phosphate and erythrose-4-phosphate. In many methanotrophs, methane is assimilated into fructose-6-phosphate. As depicted in FIG. 15, expression of phosphoketolase by microorganisms such as methanotrophic bacteria may bypass pyruvate decarboxylation and convert some or all of available fructose-6-phospate to intermediates of the non-oxidative pentose phosphate pathway and acetyl-phosphate. Such a modification may increase the carbon conversion efficiency in methanotrophic biocatalysis.

Exemplary phosphoketolases include PktA and PktB from bacteria from the genus Methylomicrobium. The nucleotide and amino acid sequences for PktA and PktB from M. buryatense, for example, are provided as SEQ ID NOS:21 and 22 (PktA) and SEQ ID NOS:23 and 24 (PktB), respectively. Functional homologs of these enzymes from other species are also suitable for use in the present disclosure. Expression may be constitutive or may be inducible to allow a convenient means to switch between the conventional Embden-Meyerhof-Parnas (EMP) pathway and the phosphoketolase-mediated non-oxidative pentose phosphate pathway (see FIG. 15).

Cells may be cultured using conventional techniques and media that will vary with the cell type. Methanotrophic cells can be cultured in either methanol or methane at a wide range of temperatures depending on the nature of the cells. Examples include temperatures ranging from 20° C. to 65° C., from 30° C. to 37° C., or temperatures greater than 37° C. or greater than 45° C. Methanol may be added directly to the medium at 0.1%-5%. Gases such as methane may be bubbled into the liquid medium continuously, or added into the headspace of sealed vials. Gases may be methane, methane/air mixtures or an array of biogas or natural gas streams that may or may not be mixed with air or varying concentrations of oxygen. For example, methanotrophic cells may be cultured in NMS2 medium at 30° C. with orbital shaking at 175 rpm. Strains may be grown in sealed 1 L glass serum bottles with 25% methane in air, or 500 mL baffled flasks supplemented with 1% methanol. Additional cultivation techniques may also be suitable, including growth in solid media or large scale fermenters.

In order to facilitate regulated, heterologous gene expression in methanotrophs, an inducible, broad-host range vector, pCAH01 (FIG. 12), was constructed by fusing the tetracycline promoter/operator (tetp/o or Ptet) from pASK75 with the IncP-based origin of the pAWP78 vector that can be replicated by Methylomicrobium spp. Experiments using GFP fluorescence as a readout of promoter activity indicated tightly controlled tetp/o-mediated gene expression in M. buryatense after induction with sub-lethal concentrations of the anhydrotetracycline inducer. The tetp/o did not show any leaky gene expression in the absence of inducer, making it an excellent tool for conditional gene expression/knock-out studies in methanotrophic bacteria that replicate vectors containing the IncP-based origin of replication.

In certain embodiments, a nucleic acid may be identical to the sequence represented herein. In other embodiments, the nucleic acids may be least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence presented herein, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence presented herein. Sequence identity calculations can be performed using computer programs, hybridization methods, or calculations. Exemplary computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, BLASTN, BLASTX, TBLASTX, and FASTA. The BLAST programs are publicly available from NCBI and other sources. For example, nucleotide sequence identity can be determined by comparing query sequences to sequences in publicly available sequence databases (NCBI) using the BLASTN2 algorithm.

The nucleic acid molecules exemplified herein encode polypeptides with amino acid sequences represented herein. In certain embodiments, the polypeptides may be at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the reference amino acid sequence while possessing the function. The present disclosure encompasses cells that contain the nucleic acid molecules described herein, have genetic modifications to the nucleic acid molecules, or express the polypeptides described herein.

“Nucleic acid” or “polynucleotide” as used herein refers to purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotide or mixed polyribo-polydeoxyribonucleotides. This includes single- and double-stranded molecules (i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids) as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases.

Nucleic acids referred to herein as “isolated” are nucleic acids that have been removed from their natural milieu or separated away from the nucleic acids of the genomic DNA or cellular RNA of their source of origin (e.g., as it exists in cells or in a mixture of nucleic acids such as a library), and may have undergone further processing. Isolated nucleic acids include nucleic acids obtained by methods described herein, similar methods or other suitable methods, including essentially pure nucleic acids, nucleic acids produced by chemical synthesis, by combinations of biological and chemical methods, and recombinant nucleic acids that are isolated. In certain embodiments, the nucleic acids are complementary DNA (cDNA) molecules.

The nucleic acids described herein may be used in methods for production of organic acids through incorporation into cells, tissues, or organisms. In some embodiments, a nucleic acid may be incorporated into a vector for expression in suitable host cells. Alternatively, gene-targeting or gene-deletion vectors may also be used to disrupt or ablate a gene. The vector may then be introduced into one or more host cells by any method known in the art. One method to produce an encoded protein includes transforming a host cell with one or more recombinant nucleic acids (such as expression vectors) to form a recombinant cell. The term “transformation” is generally used herein to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell, but can be used interchangeably with the term “transfection.”

Suitable vectors for gene expression may include (or may be derived from) plasmid vectors that are well known in the art, such as those commonly available from commercial sources. Vectors can contain one or more replication and inheritance systems for cloning or expression, one or more markers for selection in the host, and one or more expression cassettes. The inserted coding sequences can be synthesized by standard methods, isolated from natural sources, or prepared as hybrids. Ligation of the coding sequences to transcriptional regulatory elements or to other amino acid encoding sequences can be carried out using established methods. A large number of vectors, including algal, bacterial, yeast, and mammalian vectors, have been described for replication and/or expression in various host cells or cell-free systems, and may be used with genes encoding the enzymes described herein for simple cloning or protein expression.

Certain embodiments may employ promoters or regulatory operons. The efficiency of expression may be enhanced by the inclusion of enhancers that are appropriate for the particular cell system that is used, such as those described in the literature. Suitable promoters also include inducible promoters. Expression systems for constitutive expression in cells include, for example, the vectors described in the figures. Inducible expression systems are also suitable for use.

Host cells can be transformed, transfected, or infected as appropriate with gene-disrupting constructs or plasmids (e.g., an expression plasmid) by any suitable method including electroporation, calcium chloride-, lithium chloride-, lithium acetate/polyethylene glycol-, calcium phosphate-, DEAE-dextran-, liposome-mediated DNA uptake, spheroplasting, injection, microinjection, microprojectile bombardment, phage infection, viral infection, or other established methods. Alternatively, vectors containing a nucleic acid of interest can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, for example, by injection. Exemplary embodiments include a host cell or population of cells expressing one or more nucleic acid molecules or expression vectors described herein (for example, a genetically modified microorganism). The cells into which nucleic acids have been introduced as described above also include the progeny of such cells.

Vectors may be introduced into host cells by direct transformation, in which DNA is mixed with the cells and taken up without any additional manipulation, by conjugation, electroporation, or other means known in the art. Expression vectors may be expressed by host cells episomally or the gene of interest may be inserted into the chromosome of the host cell to produce cells that stably express the gene with or without the need for selective pressure. For example, expression cassettes may be targeted to neutral chromosomal sites by double recombination.

Host cells with targeted gene disruptions or carrying an expression vector (i.e., transformants or clones) may be selected using markers depending on the mode of the vector construction. The marker may be on the same or a different DNA molecule. In prokaryotic hosts, the transformant may be selected, for example, by resistance to ampicillin, tetracycline or other antibiotics. Production of a particular product based on temperature sensitivity may also serve as an appropriate marker.

In exemplary embodiments, the host cell may be a microbial cell, such as a bacterial cell or a yeast cell, and may be from any genera or species of microorganism that is known to consume C1 substrates and is genetically manipulable. Exemplary microorganisms include, but are not limited to, bacteria; fungi; archaea; protists; eukaryotes, such as algae; and animals such as plankton, planarian, and amoeba.

Host cells may be cultured in an appropriate fermentation medium. An appropriate, or effective, fermentation medium refers to any medium in which a host cell, including a genetically modified microorganism, when cultured, is capable of growing and producing products such as organic acids. Such a medium is typically an aqueous medium comprising assimilable carbon, nitrogen and phosphate sources, but can also include appropriate salts, minerals, metals and other nutrients. Microorganisms and other cells can be cultured in conventional fermentation bioreactors or photobioreactors and by any fermentation process, including batch, fed-batch, cell recycle, and continuous fermentation. The pH of the fermentation medium is regulated to a pH suitable for growth of the particular organism. Culture media and conditions for various host cells are known in the art. A wide range of media for culturing cells, for example, are available from ATCC.

Isolation or extraction of organic acids from the cells may be aided by mechanical processes such as crushing, for example, with an expeller or press, by supercritical fluid extraction, pH-induced precipitation, or the like. Once the organic acids have been released from the cells, they can be recovered or separated from a slurry of debris material (such as cellular residue, by-products, etc.). This can be done, for example, using techniques such as sedimentation or centrifugation. Recovered organic acids can be collected and directed to a conversion process if desired.

Processes for producing an organic acid may also comprise a removing step, wherein a portion of the organic acid is removed from the mixture to form substantially a pure organic acid. In some embodiments, the removing step may comprise a two-step process, wherein the first step comprises an adorption process wherein the organic acid is not adsorbed, and other components are selectively adsorbed. In some embodiments of the present invention, a first step comprising adorption will result in a substantially pure organic acid stream.

The first step may be followed by a second step polishing step. The second step may comprise crystallization. In still further embodiments of the present invention, the removing step is at least one of affinity chromatography, ion exchange chromatography, solvent extraction, liquid-liquid extraction, distillation, filtration, centrifugation, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, chromatofocusing, differential solubilization, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, or countercurrent distribution, or combinations thereof. In some embodiments of the present invention, the removing step may be performed either during the mixing step or subsequent to the mixing step or both.

Separation/purification operations may be used to generate an organic acid free of or substantially free of a wide variety of impurities that may be introduced during the biological production of an organic acid. These impurities may include fermentation salts, nutrients and media to support growth, unconverted substrate, extracellular proteins and lysed cell contents, as well as the buildup of non-target metabolites. Accumulation of these constituents in culture broth may vary greatly depending on the microorganism, substrate used for conversion, biological growth conditions and bioreactor design, and broth pretreatment.

Cell removal from the broth may be achieved by a variety of solid removal unit operations. Some examples include filtration, centrifugation, and combinations thereof. Once the microorganism cell matter has been removed, further impurity removal operations may be utilized such as exposing the mixture to activated carbon.

EXAMPLES Example 1 Plasmid and Strain Construction

Plasmids for heterologous gene expression were constructed using 2× Gibson Assembly Mix from New England Biolabs (Ipswich, Mass.) following the manufacturer's protocol. Polymerase chain reactions were performed using Q5 High-Fidelity Polymerase from New England Biolabs and primers from Integrated DNA Technologies (Coralville, Iowa). Final constructs were confirmed by sequence analysis (Genewiz, South Plainfield, N.J.). Plasmid constructs were transformed into M. alcaliphilum via conjugation. Positive transformants selected on P agar containing 100 μg/mL of kanamycin were confirmed using plasmid-specific primers in polymerase chain reactions. Escherichia coli Zymo 5a (Zymo Research, Irvine, Calif.) was used for cloning and plasmid propagation, and E. coli S17-1 λpir was used as the conjugation donor strain. E. coli strains were grown at 37° C. in Luria-Bertani (LB) broth supplemented with 50 ug/mL of kanamycin.

Cultivation, Growth Parameters, and Bioreactor Fermentations

Methylomicrobium alcaliphilum 20Z were routinely cultured in NMS2 medium (3% NaCl) at 30° C. with orbital shaking at 225 rpm. Strains were grown in sealed 150 mL glass serum bottles (Kimble Chase, Vineland, N.J.) with 20% (v/v) CH₄ in air, or 500 mL baffled flasks supplemented with 1% CH₃OH (v/v).

Batch CH₄ fermentations were performed in a 0.5 L or 5.0 L BioFlo batch bioreactor (New Brunswick Scientific, Edison, N.J.) containing NMS2 medium supplemented with 8×KNO3, 2× phosphate buffer, and 4× trace element solution to support high cell growth. The temperature was maintained at 30° C., and mixing was achieved by using a bottom marine impeller and mid-height Rushton impeller at 500 rpm. A continuous flow rate of 300 ccm (0.5 L) or 3000 ccm (5.0 L) 20% (v/v) methane in air was maintained. Antifoam was added as needed.

Off-Gas Analysis and Detection of Organic Acids

CH₄ and CO₂ off-gas was measured every 20 minutes for the duration of bioreactor fermentations by using infrared-based BlueSens gas detectors specific for CH₄ and CO₂ (Herten, Germany). CH₄ consumption was determined by calculating the difference between off-gas detected before and after inoculation. Percentage CH₄ consumption was converted to weight based on CH₄ density and the flow rate (56.64 g CH₄/24 hours, 0.5 L reactor; 566.4 g CH₄/24 hours, 5.0 L reactor).

High pressure liquid chromatography (HPLC) was used to detect excreted products in culture supernatants. At the indicated time, the OD₆₀₀ was measured and a 1 mL sample was taken for HPLC analysis. Culture supernatant was filtered using a 0.2 μm syringe filter or 0.5 mL 10K MWCO centrifuge tube (Life Technologies) and then separated using a model 1260 HPLC (Agilent, Santa Clara, Calif.) and a Rhenomonex Rezex RFQ-Fast Fruit H+ column (Bio-Rad). A 0.1 mL injection volume was used in 0.01 N sulfuric acid with a 1 mL/minute flow rate at 85° C. Refractive index and diode array detectors were used for compound detection, which were confirmed to have matching UV spectral profiles as pure compounds. Concentrations were calculated by regression analysis compared to known standards. FIG. 13 shows the production levels of cis,cis-muconic acid from a culture of Methylomicrobium alcaliphilum 20Z engineered to express the AroZ and AroY genes from K. variicola along with the CatA gene from Acinetobacter spp. (P76A variant).

Example 2 Expression of the Autotrophic Pathway

The cbbL, cbbS, and cbbQ genes encoding the subunit and regulatory proteins of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the phosphoribulokinase-encoding cbbP gene from Methylococcus capsulatus were cloned into the inducible expression vector pCAH01 to create pRubisco (FIG. 19).

The pRubisco vector has been transformed into Methylomicrobium alcaliphilum cells via biparental mating and confirmed via PCR analysis. Heterologous expression of Rubisco and phosphoribulokinase was confirmed by SDS-PAGE analysis.

The Examples discussed above are provided for purposes of illustration and are not intended to be limiting. Still other embodiments and modifications are also contemplated.

While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope. 

We claim:
 1. An engineered cell, comprising an exogenously added gene encoding a 3-dehydroshikimate dehydratase (AroZ), a protocatechuic acid decarboxylase (AroY), a catechol 1,2-dioxygenase (CatA), a phospho-2-dehydro-3-deoxyheptonate aldolase (AroG), an anthranilate synthase (TrpE), a phosphoenolpyruvate synthase, or a transketolase; wherein the engineered cell is a methanotroph and is able to convert a C1 substrate to a muconic acid and the cell comprises genes encoding the 3-dehydroshikimate dehydratase (AroZ), the protocatechuic acid decarboxylase (AroY), and the catechol 1,2-dioxygenase (CatA).
 2. The engineered cell of claim 1, wherein the 3-dehydroshikimate dehydratase (AroZ), is from Klebsiella variicola.
 3. The engineered cell of claim 1, wherein the protocatechuic acid decarboxylase (AroY) is from Enterobacter cloacae.
 4. The engineered cell of claim 1, wherein the catechol 1,2-dioxygenase (CatA) is from Acinetobacter.
 5. The engineered cell of claim 1, wherein the 3-dehydroshikimate dehydratase (AroZ) is from Klebsiella variicola, the protocatechuic acid decarboxylase (AroY) is from Enterobacter cloacae, and the catechol 1,2-dioxygenase (CatA) is from Acinetobacter.
 6. The engineered cell of claim 1, wherein the cell further comprises an exogenously added gene encoding a phosphoketolase.
 7. The engineered cell of claim 1, wherein the cell further comprises an exogenously added gene encoding a CbbL, CbbS, CbbQ or CbbP polypeptide.
 8. The engineered cell of claim 7, wherein the cell further comprises exogenously added genes encoding CbbL, CbbS, CbbQ and CbbP polypeptides.
 9. The engineered cell of claim 1, wherein the cell is from an organism from the genus Methylococcus.
 10. The engineered cell of claim 9, wherein the cell is Methylococcus capsulatus.
 11. The engineered cell of claim 1, wherein the C1 substrate is methane, methanol, carbon dioxide, carbon monoxide, syngas, natural gas, or biogas.
 12. A method for producing a muconic acid, comprising: a) culturing the engineered cell of claim 1 with a C1 substrate; and b) recovering the muconic acid from the culture. 